The first step of gene expression is the association of RNA polymerase with the promoter DNA to initiate synthesis of an RNA molecule. A molecular genetic approach is being used to determine how the eubacterial RNA polymerase recognizes the promoter DNA sequence. Previous work strongly suggests that the two domains of the promoter directly contact two domains of the sigma subunit of RNA polymerase. Cassette mutagenesis will be used to further probe the involvement of one of these domains (region 2) of sigma70, the major sigma protein of E. coli. The aims of this analysis are: (1) to determine which amino-acid residues of region 2 are essential for sigma70 activity; and (2) to obtain mutations that change the specificity of the protein for individual base-pairs in the -10 region of the promoter. Some weak promoters depend on specific positive-control proteins (activators) that bind in or near the promoter and enhance the interaction between the promoter and RNA polymerase. The current model for the mechanism of positive control is that the DNA-bound activator makes a favorable protein-protein contact with RNA polymerase. A genetic approach is planned to determine which subunit(s) of RNA polymerase are involved in this favorable interaction. Two kinds of mutant polymerases will be sought: (1) those that restore interaction with a mutant activator; and (2) those that fail to respond to a wild-type activator. This analysis will focus on two of the best-characterized activators, the cI and cII proteins of bacteriophage lambda and related phages. Another project is the analysis of the phage P22 genes that alter the O-antigen portion of the lipopolysaccharide layer of the host cell surface. This phage-encoded modification undergoes phase variation (switching on and off). Mechanisms of phase variation in enteric bacteria have been studied in great detail for proteinaceous cell-surface antigens such as flagella and pili, but the mechanism of phase variation of polysaccharide antigens is currently unknown.